Citations to some documents may be indicated as numbers in parentheses; those numbers refer to the bibliography under the heading “Related Art” at the end of this section. Those references, as well as others cited in this document are hereby incorporated by reference.
Live bacterial vaccine vectors have been used successfully to elicit effective immune responses in order to prevent infection. Such vectors have been used to induce protective immunity against infection from homologous and heterologous bacterial strains. Live attenuated bacterial vectors are also useful for food safety, for example to prevent or reduce infection of livestock animals such as poultry or cattle by bacterial strains that are pathogenic to humans, such as Salmonella or E. coli. 
The ability of live attenuated pathogenic bacteria of the Enterobacteriaceae family to colonize the gut-associated lymphoid tissue (GALT; Peyer's patches) and the deep tissues following oral administration is beneficial in that it stimulates all arms of the immune response, including mucosal, humoral and cellular immunities (Curtiss/Doggett/Nayak/Srinivasan 1996; Galan and Sansonetti 1996; Medina/Guzman 2001). Colonization of the intestinal tract by gram negative bacteria is dependent in part upon the expression of a number of surface antigens, including LPS O-antigen side chains, a diversity of fimbrial adhesins, flagella and certain outer membrane proteins. Thus, rough mutants, i.e., those with little or no O-antigen on their LPS, that have mutational lesions precluding synthesis of LPS O-antigen or parts of the LPS core tend not to colonize the intestinal tract (Roantree, 1971; Nnalue, 1990) and are defective in attaching to and invading intestinal cells and surviving in cells on the other side of the intestinal wall barrier. (25, 26). This latter phenotype is due to the fact that LPS is needed for bacteria to display resistance to killing by macrophages (27, 28) and also for the display of serum resistance (29, 30), that is, the ability to multiply in blood. In accord with these observations, rough mutants defective in LPS synthesis and thus defective in infection are among the most frequently isolated using signature tagged mutagenesis (31) and genes for LPS biosynthesis are very often up-regulated during infection as revealed by use of in vivo expression technology (32). Rough mutants have generally not been very effective when used as live vaccines. (33, 34, Hill abstract). Thus, it follows that an attenuated immunogenic live bacterial vaccine, to be safe and efficacious must not only display avirulence and not induce disease symptomology, but also must be able to reach, multiply and persist for a while in those lymphoid organs necessary to stimulate a protective immune response. Permanently rough strains cannot achieve the latter. The use of bacterial strains with mutations in the galE locus encoding UDP-galactose epimerase, an enzyme that interconverts UDP-glucose and UDP-galactose (UDP-gal) (35), has been considered as a way of overcoming the above limitation. UDP-gal is needed for the synthesis of both the LPS core and O-antigen in many bacterial strains. (36). When Salmonella galE mutants are provided low levels of galactose, they make normal LPS, but when deprived of galactose, they rapidly lose the ability to synthesize a complete LPS O-antigen and core. (37). One of the difficulties with galE mutants is that they are exceedingly sensitive to galactose (38, 39) and accumulate Gal-resistant mutants that are permanently rough and therefore not only avirulent, but also non immunogenic. Because of the LPS core defect, these galE mutants are somewhat hyper attenuated and do not induce high-level protective immunity. (40, 41). Another alternative to generate a reversibly rough phenotype is to make use of pmi mutants that have a mutation in the gene for phosphomannose isomerase (42), which interconverts mannose 6-phosphate and fructose 6-phosphate. Mannose 6-phosphate is then converted to GDP-mannose which is used for synthesis of O-antigen side chains (43). pmi mutants are not mannose sensitive and, as shown by Collins et al. (44), are attenuated and somewhat immunogenic. pmi mutants, when grown in media containing mannose, synthesize wild-type levels of LPS O-antigen side chains. In addition, pmi mutants do not lose the ability to synthesize LPS core.
Immune responses to iron-regulated outer membrane proteins (IROMPS) are known to be effective in preventing septicemic infection with enteropathogens. (Bolin 1987). Further, many bacterial serotypes and species in the Enterobacteriaceae family synthesize IROMPs and other proteins involved in iron uptake that share significant antigenic homology such that antibodies induced to proteins from one bacterial serotype or species are effective in binding to IROMPS and other iron uptake proteins from other serotypes and species. (Jun Lin 2001).
The fur gene encodes a repressor that represses all genes encoding IROMPS, in the presence of free iron. (Earhart 1996). When iron concentrations become low, as is the case in most animal host tissues beyond the intestinal wall barrier, the fur repression decreases and higher level expression of IROMPS and other fur-regulated genes needed to sequester iron is observed. fur mutants are attenuated when fed orally, giving a two to three log higher LD50 when administered either to mice (52) or day-of-hatch chicks. On the other hand, administering a fur mutant of S. typhimurium by the intraperitoneal route leads to only a slightly elevated LD50 compared to that of the wild-type parent. (53). In the intestinal tract iron is plentiful, both due to non absorption of dietary iron and the presence of iron from hemoglobin breakdown contributed into the intestinal tract as a component of bile. Green et al. 1968. It is also well known that iron, unless in a complex form, can promote the formation of damaging hydroxyl radicals, which may account, in part, for the toxicity of iron (51). Thus the high oral LD50 of fur mutants may be due to toxicity of free iron encountered in the intestinal tract. fur mutants are also acid sensitive (55) and are thus potentially sensitive to the gastric acidity barrier and to killing in acidified phagosomes in macrophages (56, 57). In summary, while fur mutant bacterial strains would display higher levels of IROMPs that likely would induce protective immunity, their avirulence properties when administered orally make them poor immunogens. So, while mutants unable to produce Fur are attenuated when delivered orally, because of substantial iron induced death they do not induce a significant immune response.
Members of the Enterobacteriaceae family cause a wide variety of human and animal diseases, including gram-negative sepsis, food poisoning, and typhoid fever. In addition, many farm animals are colonized with diverse enteric bacteria such as many serotypes of Salmonella without causing disease. Such bacteria are capable of transmission through the food chain to cause diseases in humans. Developing vaccines to prevent all the types of enteric diseases caused by bacterial enteric pathogens of diverse genera, species and serotypes and to prevent colonization by these diverse bacterial types in farm animals to enhance food safety would be prohibitively expensive. The incidence of these diseases and the prevalence of colonization of farm animals highlights the need for vaccines that would cross-protect against the numerous species and serotypes of enteric bacteria. Thus, it would be useful to develop attenuated bacterial vaccine strains that are capable of inducing cross-protective immunity.